Production of steviol glycosides in recombinant hosts

Abstract

The invention relates to recombinant microorganisms and methods for producing steviol glycosides and steviol glycoside precursors.

What is claimed is: 
     
       1. A recombinant host cell producing a steviol glycoside in a cell culture, wherein the host cell comprises a recombinant gene encoding a Sugar Efflux Transporter (SET) polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:18;
 wherein the recombinant host cell expresses exogenous nucleic acids encoding: 
 (a) a polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP); 
 (b) a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP; 
 (c) a polypeptide capable of synthesizing ent-kaurenoic acid from ent-kaurene; 
 (d) a polypeptide capable of synthesizing ent-kaurene from ent-copalyl pyrophosphate; 
 (e) a polypeptide capable of synthesizing steviol from ent-kaurenoic acid; 
 (f) a polypeptide capable of reducing cytochrome P450 complex; and 
 (g) one or more uridine diphosphate (UDP) glycosyltransferases (UGTs) polypeptide; and 
 wherein the host cell is a plant cell, a fungal cell, or a bacterial cell. 
 
     
     
       2. The host cell of  claim 1 , wherein the host cell further comprises a deletion of a gene encoding a trafficking adapter polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:46. 
     
     
       3. The host cell of  claim 2 , wherein the deletion of the gene encoding the polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:46 increases excretion of Rebaudioside D (RebD). 
     
     
       4. The host cell of  claim 2 , wherein the deletion of the gene encoding the polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:48 increases excretion of Rebaudioside M (RebM). 
     
     
       5. The host cell of  claim 1 , wherein the host cell further comprises a deletion of a gene encoding a transporter polypeptide having the amino acid sequence set forth in SEQ ID NO:102 or a functional homolog thereof. 
     
     
       6. The recombinant host cell of  claim 1 , wherein:
 (a) the polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) comprises a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:182; 
 (b) the polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP comprises a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:184; 
 (c) the polypeptide capable of synthesizing ent-kaurenoic acid from ent-kaurene comprises a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:188; 
 (d) the polypeptide capable of synthesizing ent-kaurene from ent-copalyl pyrophosphate comprises a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:186; 
 (e) the polypeptide capable of synthesizing steviol from ent-kaurenoic acid comprises a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:192; 
 (f) the polypeptide capable of reducing cytochrome P450 complex comprises a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:194; and wherein the one or more uridine diphosphate (UDP) glycosyltransferases (UGTs) polypeptide comprises: 
 (g) a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group;
 wherein the polypeptide comprises a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:196; 
 
 (h) a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside;
 wherein the polypeptide comprises a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 200; 
 
 (i) a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group;
 wherein the polypeptide comprises a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:198; and/or 
 
 (j) a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside;
 wherein the polypeptide comprises a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:204. 
 
 
     
     
       7. The host cell of  claim 1 , wherein the bacterial cell comprises  Escherichia  bacteria cells,  Lactobacillus  bacteria cells,  Lactococcus  bacteria cells,  Cornebacterium  bacteria cells,  Acetobacter  bacteria cells,  Acinetobacter  bacteria cells, or  Pseudomonas  bacterial cells. 
     
     
       8. The host cell of  claim 1 , wherein the fungal cell is a yeast cell. 
     
     
       9. The host cell of  claim 8 , wherein the yeast cell is a cell from  Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous , or  Candida albicans  species. 
     
     
       10. The host cell of  claim 5 , wherein the host cell excretes a decreased amount of steviol-13-O-glucoside (13-SMG) relative to a steviol glycoside-producing host cell that does not have the deletion of the one or more genes encoding the one or more transporter polypeptides. 
     
     
       11. The host cell of  claim 5 , wherein the host cell excretes an increased amount of RebA, RebB, RebD, and/or RebM relative to a steviol glycoside-producing host cell that does not have the deletion of the one or more genes encoding the one or more transporter polypeptides. 
     
     
       12. The host cell of  claim 5 , wherein the host cell produces an increased amount of RebA, RebB, RebD, and/or RebM relative to a steviol glycoside-producing host cell that does not have the deletion of the one or more genes encoding the one or more transporter polypeptides. 
     
     
       13. A method of increasing production of a steviol glycoside in a recombinant host cell or increasing excretion of a steviol glycoside into a culture medium, comprising culturing the host cell of  claim 1  in a cell culture, under conditions in which one or more of the genes are expressed;
 wherein the steviol glycoside is produced by the host cell. 
 
     
     
       14. The method of  claim 13 , wherein the steviol glycoside is RebA, RebB, RebD, and/or RebM. 
     
     
       15. A method of increasing an amount of RebA, RebB, RebD, and/or RebM produced by a recombinant host cell, comprising culturing the host cell of  claim 1  in a cell culture;
 wherein excretion of 13-SMG from the host cell into the cell culture is decreased relative to RebA, RebB, RebD, and/or RebM-producing host cell that does not have the deletion of the one or more genes encoding the one or more transporter polypeptides; 
 wherein RebA, RebB, RebD, and/or RebM are produced by the host cell. 
 
     
     
       16. The method of  claim 13 , that further comprises isolating RebA, RebB, RebD, and/or RebM, alone or in combination from the cell culture. 
     
     
       17. The method of  claim 16 , wherein the isolating step comprises separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising RebA, RebB, RebD, and/or RebM, alone or in combination, and:
 (a) contacting the supernatant with one or more adsorbent resins in order to obtain at least a portion of RebA, RebB, RebD, and/or RebM, alone or in combination; or 
 (b) contacting the supernatant with one or more ion exchange or reversed-phase chromatography columns in order to obtain at least a portion of RebA, RebB, RebD, and/or RebM, alone or in combination; or 
 (c) crystallizing or extracting RebA, RebB, RebD, and/or RebM, alone or in combination; 
 thereby isolating RebA, RebB, RebD, and/or RebM, alone or in combination. 
 
     
     
       18. The method of  claim 13 , that further comprises recovering RebA, RebB, RebD, and/or RebM alone or a composition comprising RebA, RebB, RebD, and/or RebM from the cell culture. 
     
     
       19. The method of  claim 18 , wherein the recovered composition is enriched for RebA, RebB, RebD, and/or RebM, relative to a steviol glycoside composition of  Stevia  plant and has a reduced level of  Stevia  plant-derived components relative to a steviol glycoside composition obtained from a plant-derived  Stevia  extract. 
     
     
       20. The method of  claim 13 , wherein the cell culture comprises:
 (a) RebA, RebB, RebD, and/or RebM produced by the host cell of  claim 1 , 
 (b) glucose, fructose, sucrose, xylose, rhamnose, uridine diphosphate (UDP)-glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine; and 
 (c) supplemental nutrients comprising trace metals, vitamins, salts, yeast nitrogen base (YNB), and/or amino acids. 
 
     
     
       21. A cell culture, comprising the host cell of  claim 1 , the cell culture further comprising:
 (a) RebA, RebB, RebD, and/or RebM produced by the host cell; 
 (b) glucose, fructose, sucrose, xylose, rhamnose, uridine diphosphate (UDP)-glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine; and 
 (c) supplemental nutrients comprising trace metals, vitamins, salts, YNB, and/or amino acids; 
 wherein RebA, RebB, RebD, and/or RebM are present at a concentration of at least 1 mg/liter of the cell culture. 
 
     
     
       22. A cell lysate, comprising RebA, RebB, RebD, and/or RebM produced by the host cell of  claim 1 , and the cell lysate further comprising glucose, fructose, sucrose, xylose, rhamnose, uridine diphosphate (UDP)-glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine, supplemental nutrients comprising trace metals, vitamins, salts, YNB, and/or amino acids. 
     
     
       23. The host cell of  claim 1 , wherein the host cell is a  Yarrowia lipolytica  cell. 
     
     
       24. The method of  claim 13 , wherein:
 (a) Reb A is produced in the recombinant host cell expressing the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; the polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-0-glucose of a steviol glycoside; the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; 
 (b) Reb B is produced in the recombinant host cell expressing the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; the polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-0-glucose of a steviol glycoside; and the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; 
 (c) Reb D is produced in the recombinant host cell expressing the polypeptide capable of glycosylating steviol or the steviol glycoside at its C-13 hydroxyl group; the polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-0-glucose of a steviol glycoside; the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; and/or 
 (d) Reb M is produced in the recombinant host cell expressing the polypeptide capable of glycosylation of the 13-OH of steviol; the polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside. 
 
     
     
       25. The method of  claim 13 , wherein Reb A is produced in the recombinant host cell expressing the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; the polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside. 
     
     
       26. The method of  claim 13 , wherein Reb D is produced in the recombinant host cell expressing the polypeptide capable of glycosylating steviol or the steviol glycoside at its C-13 hydroxyl group; the polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside. 
     
     
       27. The method of  claim 13 , wherein Reb M is produced in the recombinant host cell expressing the polypeptide capable of glycosylation of the 13-OH of steviol; the polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside. 
     
     
       28. The recombinant host of  claim 1 , wherein the steviol glycoside is Rebaudioside A (Reb A), Rebaudioside B (Reb B), Reb D and/or Reb M or an isomer thereof.